U.S. patent application number 17/059999 was filed with the patent office on 2021-07-22 for over-expression of transcriptional activator/repressor gis1 in yeast for increased ethanol production.
The applicant listed for this patent is DANISCO US INC. Invention is credited to Daniel Joseph MACOOL, Paula Johanna Maria TEUNISSEN, Quinn Qun ZHU.
Application Number | 20210221857 17/059999 |
Document ID | / |
Family ID | 1000005521674 |
Filed Date | 2021-07-22 |
United States Patent
Application |
20210221857 |
Kind Code |
A1 |
MACOOL; Daniel Joseph ; et
al. |
July 22, 2021 |
OVER-EXPRESSION OF TRANSCRIPTIONAL ACTIVATOR/REPRESSOR GIS1 IN
YEAST FOR INCREASED ETHANOL PRODUCTION
Abstract
Described are compositions and methods relating to modified
yeast cells that over-expresses the transcriptional
activator/repressor, GIS1. The yeast cells produce an increased
amount of ethanol compared to their parental cells. Such yeast is
particularly useful for large-scale ethanol production from starch
substrates where increased ethanol production is desirable
Inventors: |
MACOOL; Daniel Joseph;
(RUTLEDGE, PA) ; TEUNISSEN; Paula Johanna Maria;
(Palo Alto, CA) ; ZHU; Quinn Qun; (WEST CHESTER,
PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DANISCO US INC |
Palo Alto |
CA |
US |
|
|
Family ID: |
1000005521674 |
Appl. No.: |
17/059999 |
Filed: |
May 21, 2019 |
PCT Filed: |
May 21, 2019 |
PCT NO: |
PCT/US2019/033207 |
371 Date: |
November 30, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62677888 |
May 30, 2018 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/395 20130101;
C07K 14/39 20130101; C12P 7/06 20130101 |
International
Class: |
C07K 14/395 20060101
C07K014/395; C07K 14/39 20060101 C07K014/39; C12P 7/06 20060101
C12P007/06 |
Claims
1. Modified yeast cells derived from parental yeast cells, the
modified cells comprising a genetic alteration that causes the
modified cells to produce an increased amount of GIS1 polypeptides
compared to the parental cells, wherein the modified cells produce
during fermentation an increased amount of alcohol compared to the
amount of alcohol produced by the parental cells under identical
fermentation conditions.
2. The modified cells of claim 1, wherein the genetic alteration
comprises the introduction into the parental cells of a nucleic
acid capable of directing the expression of a GIS1 polypeptide to a
level above that of the parental cell grown under equivalent
conditions.
3. The modified cells of claim 1, wherein the genetic alteration
comprises the introduction of an expression cassette for expressing
a GIS1 polypeptide.
4. The modified cells of claim 1, wherein the genetic alteration
comprises the introduction of an exogenous YDR096W gene.
5. The modified cells of claim 2, wherein the genetic alteration
comprises the introduction of a stronger promoter in an endogenous
YDR096W gene.
6. The modified cells of any of claims 1-5, wherein the amount of
increase in the expression of the GIS1 polypeptide is at least
about 10-fold compared to the level expression in the parental
cells grown under equivalent conditions.
7. The modified cells of any of claims 1-5, wherein the amount of
increase in the production of mRNA encoding the GIS1 polypeptide is
at least about 10-fold compared to the level in the parental cells
grown under equivalent conditions.
8. The modified cells of any of claims 1-7, wherein the cells
further comprise an exogenous gene encoding a carbohydrate
processing enzyme.
9. The modified cells of any of claims 1-8, further comprising an
alteration in the glycerol pathway and/or the acetyl-CoA
pathway.
10. The modified cells of any of claims 1-9, further comprising an
alternative pathway for making ethanol.
11. The modified cells of any of claims 1-10, wherein the cells are
of a Saccharomyces spp.
12. A method for increasing the production of ethanol in yeast
cells grown on a carbohydrate substrate, comprising: introducing
into parental yeast cells a genetic alteration that causes the
modified cells to produce an increased amount of GIS1 polypeptides
compared to the parental cells, wherein the modified cells produce
an increased amount of ethanol compared to otherwise identical
parental cells under equivalent fermentation conditions.
13. The method of claim 12, wherein the genetic alteration
comprises introduction into the parental cells of a nucleic acid
capable of directing the expression of GIS1 to a level greater that
of the parental cell grown under equivalent conditions.
14. The method of claim 12, wherein the genetic alteration
comprises introduction of an expression cassette for expressing
GIS1.
15. The method of any of claims 12-14, wherein the cells further
comprise one or more genes of the phosphoketolase pathway.
16. The method of claim 15, wherein the genes of the
phosphoketolase pathway are selected from the group consisting of
phosphoketolase, phosphotransacetylase and acetylating acetyl
dehydrogenase.
17. The method of any of claims 12-16, wherein the amount of
increase in the expression of GIS1 is at least 10-fold higher
compared to the level of expression in parental cells grown under
equivalent conditions, based on GIS1-encoding mRNA expression.
18. The method of any of any of claims 12-17, wherein the cells
further comprise an exogenous gene encoding a carbohydrate
processing enzyme.
19. The method of any of claims 12-18, wherein the cells further
comprise an alteration in the glycerol pathway and/or the
acetyl-CoA pathway.
20. The method of any of the claims 12-19, wherein the cells
further comprise an alternative pathway for making ethanol.
21. The method of any of claims 12-20, wherein the cells are of a
Saccharomyces spp.
Description
TECHNICAL FIELD
[0001] The present compositions and methods relate to modified
yeast cells that over-expresses the transcriptional
activator/repressor, GIS1. The yeast cells produce an increased
amount of ethanol compared to their parental cells. Such yeast is
particularly useful for large-scale ethanol production from starch
substrates where increased ethanol production is desirable.
BACKGROUND
[0002] Yeast-based ethanol production is based on the conversion of
sugars into ethanol. The current annual fuel ethanol production by
this method is about 90 billion liters worldwide. It is estimated
that about 70% of the cost of ethanol production is the feedstock.
Since the ethanol production volume is so large, even small yield
improvements have massive economic impact for the industry. The
conversion of one mole of glucose into two moles of ethanol and two
moles of carbon dioxide is redox-neutral, with the maximum
theoretical yield being about 51%. The current industrial yield is
about 45%; therefore, there are opportunities to increase ethanol
production. Despite advances in yeast productivity, the need exists
to further modify yeast metabolic pathways to maximize ethanol
production, while not increasing the production of undesirable
by-products.
SUMMARY
[0003] The present compositions and methods relate to modified
yeast cells that over-expresses the transcriptional
activator/repressor, GIS1. The yeast cells are characterized by
increased levels of ethanol production amount compared to their
parental cells. Aspects and embodiments of the compositions and
methods are described in the following, independently-numbered,
paragraphs.
[0004] 1. In one aspect, modified yeast cells derived from parental
yeast cells are provided, the modified cells comprising a genetic
alteration that causes the modified cells to produce an increased
amount of GIS1 polypeptides compared to the parental cells, wherein
the modified cells produce during fermentation an increased amount
of alcohol compared to the amount of alcohol produced by the
parental cells under identical fermentation conditions.
[0005] 2. In some embodiments of the modified cells of paragraph 1,
the genetic alteration comprises the introduction into the parental
cells of a nucleic acid capable of directing the expression of a
GIS1 polypeptide to a level above that of the parental cell grown
under equivalent conditions.
[0006] 3. In some embodiments of the modified cells of paragraph 1,
the genetic alteration comprises the introduction of an expression
cassette for expressing a GIS1 polypeptide.
[0007] 4. In some embodiments of the modified cells of paragraph 1,
the genetic alteration comprises the introduction of an exogenous
YDR096W gene.
[0008] 5. In some embodiments of the modified cells of paragraph 2,
the genetic alteration comprises the introduction of a stronger
promoter in an endogenous YDR096W gene.
[0009] 6. In some embodiments of the modified cells of any of
paragraphs 1-5, the amount of increase in the expression of the
GIS1 polypeptide is at least about 10-fold compared to the level
expression in the parental cells grown under equivalent
conditions.
[0010] 7. In some embodiments of the modified cells of any of
paragraphs 1-5, the amount of increase in the production of mRNA
encoding the GIS1 polypeptide is at least about 10-fold compared to
the level in the parental cells grown under equivalent
conditions.
[0011] 8. In some embodiments of the modified cells of any of
paragraphs 1-7, the cells further comprise an exogenous gene
encoding a carbohydrate processing enzyme.
[0012] 9. In some embodiments, the modified cells of any of
paragraphs 1-8, further comprise an alteration in the glycerol
pathway and/or the acetyl-CoA pathway.
[0013] 10. In some embodiments, the modified cells of any of
paragraphs 1-9, further comprise an alternative pathway for making
ethanol.
[0014] 11. In some embodiments of the modified cells of any of
paragraphs 1-10, the cells are of a Saccharomyces spp.
[0015] 12. In another aspect, a method for increasing the
production of ethanol in yeast cells grown on a carbohydrate
substrate is provided, comprising: introducing into parental yeast
cells a genetic alteration that causes the modified cells to
produce an increased amount of GIS1 polypeptides compared to the
parental cells, wherein the modified cells produce an increased
amount of ethanol compared to otherwise identical parental cells
under equivalent fermentation conditions.
[0016] 13. In some embodiments of the method of paragraph 12, the
genetic alteration comprises introduction into the parental cells
of a nucleic acid capable of directing the expression of GIS1 to a
level greater that of the parental cell grown under equivalent
conditions.
[0017] 14. In some embodiments of the method of paragraph 12, the
genetic alteration comprises introduction of an expression cassette
for expressing GIS1.
[0018] 15. In some embodiments of the method of any of paragraphs
12-14, the cells further comprise one or more genes of the
phosphoketolase pathway.
[0019] 16. In some embodiments of the method of paragraph 15, the
genes of the phosphoketolase pathway are selected from the group
consisting of phosphoketolase, phosphotransacetylase and
acetylating acetyl dehydrogenase.
[0020] 17. In some embodiments of the method of any of paragraphs
12-16, the amount of increase in the expression of GIS1 is at least
10-fold higher compared to the level of expression in parental
cells grown under equivalent conditions, based on GIS1-encoding
mRNA expression.
[0021] 18. In some embodiments of the method of any of any of
paragraphs 12-17, the cells further comprise an exogenous gene
encoding a carbohydrate processing enzyme.
[0022] 19. In some embodiments of the method of any of paragraphs
12-18, the cells further comprise an alteration in the glycerol
pathway and/or the acetyl-CoA pathway.
[0023] 20. In some embodiments of the method of any of the
paragraphs 12-19, the cells further comprise an alternative pathway
for making ethanol.
[0024] 21. In some embodiments of the method of any of paragraphs
12-20, the cells are of a Saccharomyces spp.
[0025] These and other aspects and embodiments of present modified
cells and methods will be apparent from the description.
DETAILED DESCRIPTION
I. Definitions
[0026] Prior to describing the present yeast and methods in detail,
the following terms are defined for clarity. Terms not defined
should be accorded their ordinary meanings as used in the relevant
art.
[0027] As used herein, the term "alcohol" refers to an organic
compound in which a hydroxyl functional group (--OH) is bound to a
saturated carbon atom.
[0028] As used herein, the terms "yeast cells," "yeast strains," or
simply "yeast" refer to organisms from the phyla Ascomycota and
Basidiomycota. Exemplary yeast is budding yeast from the order
Saccharomycetales. Particular examples of yeast are Saccharomyces
spp., including but not limited to S. cerevisiae. Yeast include
organisms used for the production of fuel alcohol as well as
organisms used for the production of potable alcohol, including
specialty and proprietary yeast strains used to make
distinctive-tasting beers, wines, and other fermented
beverages.
[0029] As used herein, the phrase "engineered yeast cells,"
"variant yeast cells," "modified yeast cells," or similar phrases,
refer to yeast that include genetic modifications and
characteristics described herein. Variant/modified yeast do not
include naturally occurring yeast.
[0030] As used herein, the terms "polypeptide" and "protein" (and
their respective plural forms) are used interchangeably to refer to
polymers of any length comprising amino acid residues linked by
peptide bonds. The conventional one-letter or three-letter codes
for amino acid residues are used herein and all sequence are
presented from an N-terminal to C-terminal direction. The polymer
can comprise modified amino acids, and it can be interrupted by
non-amino acids. The terms also encompass an amino acid polymer
that has been modified naturally or by intervention; for example,
disulfide bond formation, glycosylation, lipidation, acetylation,
phosphorylation, or any other manipulation or modification, such as
conjugation with a labeling component. Also included within the
definition are, for example, polypeptides containing one or more
analogs of an amino acid (including, for example, unnatural amino
acids, etc.), as well as other modifications known in the art.
[0031] As used herein, functionally and/or structurally similar
proteins are considered to be "related proteins," or "homologs."
Such proteins can be derived from organisms of different genera
and/or species, or different classes of organisms (e.g., bacteria
and fungi), or artificially designed. Related proteins also
encompass homologs determined by primary sequence analysis,
determined by secondary or tertiary structure analysis, or
determined by immunological cross-reactivity, or determined by
their functions.
[0032] As used herein, the term "homologous protein" refers to a
protein that has similar activity and/or structure to a reference
protein. It is not intended that homologs necessarily be
evolutionarily related. Thus, it is intended that the term
encompass the same, similar, or corresponding enzyme(s) (i.e., in
terms of structure and function) obtained from different organisms.
In some embodiments, it is desirable to identify a homolog that has
a quaternary, tertiary and/or primary structure similar to the
reference protein. In some embodiments, homologous proteins induce
similar immunological response(s) as a reference protein. In some
embodiments, homologous proteins are engineered to produce enzymes
with desired activity(ies).
[0033] The degree of homology between sequences can be determined
using any suitable method known in the art (see, e.g., Smith and
Waterman (1981) Adv. Appl. Math. 2:482; Needleman and Wunsch (1970)
J. Mol. Biol., 48:443; Pearson and Lipman (1988) Proc. Natl. Acad.
Sci. USA 85:2444; programs such as GAP, BESTFIT, FASTA, and TFASTA
in the Wisconsin Genetics Software Package (Genetics Computer
Group, Madison, Wis.); and Devereux et al. (1984) Nucleic Acids
Res. 12:387-95).
[0034] For example, PILEUP is a useful program to determine
sequence homology levels. PILEUP creates a multiple sequence
alignment from a group of related sequences using progressive,
pair-wise alignments. It can also plot a tree showing the
clustering relationships used to create the alignment. PILEUP uses
a simplification of the progressive alignment method of Feng and
Doolittle, (Feng and Doolittle (1987) J. Mol. Evol. 35:351-60). The
method is similar to that described by Higgins and Sharp ((1989)
CABIOS 5:151-53). Useful PILEUP parameters including a default gap
weight of 3.00, a default gap length weight of 0.10, and weighted
end gaps. Another example of a useful algorithm is the BLAST
algorithm, described by Altschul et al. ((1990) J. Mol. Biol.
215:403-10) and Karlin et al. ((1993) Proc. Natl. Acad. Sci. USA
90:5873-87). One particularly useful BLAST program is the
WU-BLAST-2 program (see, e.g., Altschul et al. (1996) Meth.
Enzymol. 266:460-80). Parameters "W," "T," and "X" determine the
sensitivity and speed of the alignment. The BLAST program uses as
defaults a word-length (W) of 11, the BLOSUM62 scoring matrix (see,
e.g., Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA
89:10915) alignments (B) of 50, expectation (E) of 10, M'S, N'-4,
and a comparison of both strands.
[0035] As used herein, the phrases "substantially similar" and
"substantially identical," in the context of at least two nucleic
acids or polypeptides, typically means that a polynucleotide or
polypeptide comprises a sequence that has at least about 70%
identity, at least about 75% identity, at least about 80% identity,
at least about 85% identity, at least about 90% identity, at least
about 91% identity, at least about 92% identity, at least about 93%
identity, at least about 94% identity, at least about 95% identity,
at least about 96% identity, at least about 97% identity, at least
about 98% identity, or even at least about 99% identity, or more,
compared to the reference (i.e., wild-type) sequence. Percent
sequence identity is calculated using CLUSTAL W algorithm with
default parameters. See Thompson et al. (1994) Nucleic Acids Res.
22:4673-4680. Default parameters for the CLUSTAL W algorithm
are:
TABLE-US-00001 Gap opening penalty: 10.0 Gap extension penalty:
0.05 Protein weight matrix: BLOSUM series DNA weight matrix: IUB
Delay divergent sequences %: 40 Gap separation distance: 8 DNA
transitions weight: 0.50 List hydrophilic residues: GPSNDQEKR Use
negative matrix: OFF Toggle Residue specific penalties: ON Toggle
hydrophilic penalties: ON Toggle end gap separation penalty OFF
[0036] Another indication that two polypeptides are substantially
identical is that the first polypeptide is immunologically
cross-reactive with the second polypeptide. Typically, polypeptides
that differ by conservative amino acid substitutions are
immunologically cross-reactive. Thus, a polypeptide is
substantially identical to a second polypeptide, for example, where
the two peptides differ only by a conservative substitution.
Another indication that two nucleic acid sequences are
substantially identical is that the two molecules hybridize to each
other under stringent conditions (e.g., within a range of medium to
high stringency).
[0037] As used herein, the term "gene" is synonymous with the term
"allele" in referring to a nucleic acid that encodes and directs
the expression of a protein or RNA. Vegetative forms of filamentous
fungi are generally haploid, therefore a single copy of a specified
gene (i.e., a single allele) is sufficient to confer a specified
phenotype. The term "allele" is generally preferred when an
organism contains more than one similar genes, in which case each
different similar gene is referred to as a distinct "allele."
[0038] As used herein, the term "expressing a polypeptide" and
similar terms refers to the cellular process of producing a
polypeptide using the translation machinery (e.g., ribosomes) of
the cell.
[0039] As used herein, "over-expressing a polypeptide," "increasing
the expression of a polypeptide," and similar terms, refer to
expressing a polypeptide at higher-than-normal levels compared to
those observed with parental or "wild-type cells that do not
include a specified genetic modification. Overexpression may be
described in terms of mRNA levels or polypeptide levels, as
experimental conditions permit.
[0040] As used herein, an "expression cassette" refers to a DNA
fragment that includes a promoter, and amino acid coding region and
a terminator (i.e., promoter::amino acid coding region::terminator)
and other nucleic acid sequence needed to allow the encoded
polypeptide to be produced in a cell. Expression cassettes can be
exogenous (i.e., introduced into a cell) or endogenous (i.e.,
extant in a cell).
[0041] As used herein, the terms "fused" and "fusion" with respect
to two DNA fragments, such as a promoter and the coding region of a
polypeptide refer to a physical linkage causing the two DNA
fragments to become a single molecule.
[0042] As used herein, the terms "wild-type" and "native" are used
interchangeably and refer to genes, proteins or strains found in
nature, or that are not intentionally modified for the advantage of
the presently described yeast.
[0043] As used herein, the term "protein of interest" refers to a
polypeptide that is desired to be expressed in modified yeast. Such
a protein can be an enzyme, a substrate-binding protein, a
surface-active protein, a structural protein, a selectable marker,
a signal transducer, a receptor, a transporter, a transcription
factor, a translation factor, a co-factor, or the like, and can be
expressed. The protein of interest is encoded by an endogenous gene
or a heterologous gene (i.e., gene of interest") relative to the
parental strain. The protein of interest can be expressed
intracellularly or as a secreted protein.
[0044] As used herein, "disruption of a gene" refers broadly to any
genetic or chemical manipulation, i.e., mutation, that
substantially prevents a cell from producing a function gene
product, e.g., a protein, in a host cell. Exemplary methods of
disruption include complete or partial deletion of any portion of a
gene, including a polypeptide-coding sequence, a promoter, an
enhancer, or another regulatory element, or mutagenesis of the
same, where mutagenesis encompasses substitutions, insertions,
deletions, inversions, and combinations and variations, thereof,
any of which mutations substantially prevent the production of a
function gene product. A gene can also be disrupted using CRISPR,
RNAi, antisense, or any other method that abolishes gene
expression. A gene can be disrupted by deletion or genetic
manipulation of non-adjacent control elements. As used herein,
"deletion of a gene," refers to its removal from the genome of a
host cell. Where a gene includes control elements (e.g., enhancer
elements) that are not located immediately adjacent to the coding
sequence of a gene, deletion of a gene refers to the deletion of
the coding sequence, and optionally adjacent enhancer elements,
including but not limited to, for example, promoter and/or
terminator sequences, but does not require the deletion of
non-adjacent control elements. Deletion of a gene also refers to
the deletion a part of the coding sequence, or a part of promoter
immediately or not immediately adjacent to the coding sequence,
where there is no functional activity of the interested gene
existed in the engineered cell.
[0045] As used herein, the terms "genetic manipulation" and
"genetic alteration" are used interchangeably and refer to the
alteration/change of a nucleic acid sequence. The alteration can
include but is not limited to a substitution, deletion, insertion
or chemical modification of at least one nucleic acid in the
nucleic acid sequence.
[0046] As used herein, a "functional polypeptide/protein" is a
protein that possesses an activity, such as an enzymatic activity,
a binding activity, a surface-active property, a signal transducer,
a receptor, a transporter, a transcription factor, a translation
factor, a co-factor, or the like, and which has not been
mutagenized, truncated, or otherwise modified to abolish or reduce
that activity. Functional polypeptides can be thermostable or
thermolabile, as specified.
[0047] As used herein, "a functional gene" is a gene capable of
being used by cellular components to produce an active gene
product, typically a protein. Functional genes are the antithesis
of disrupted genes, which are modified such that they cannot be
used by cellular components to produce an active gene product, or
have a reduced ability to be used by cellular components to produce
an active gene product.
[0048] As used herein, yeast cells have been "modified to prevent
the production of a specified protein" if they have been
genetically or chemically altered to prevent the production of a
functional protein/polypeptide that exhibits an activity
characteristic of the wild-type protein. Such modifications
include, but are not limited to, deletion or disruption of the gene
encoding the protein (as described, herein), modification of the
gene such that the encoded polypeptide lacks the aforementioned
activity, modification of the gene to affect post-translational
processing or stability, and combinations, thereof
[0049] As used herein, "attenuation of a pathway" or "attenuation
of the flux through a pathway," i.e., a biochemical pathway, refers
broadly to any genetic or chemical manipulation that reduces or
completely stops the flux of biochemical substrates or
intermediates through a metabolic pathway. Attenuation of a pathway
may be achieved by a variety of well-known methods. Such methods
include but are not limited to: complete or partial deletion of one
or more genes, replacing wild-type alleles of these genes with
mutant forms encoding enzymes with reduced catalytic activity or
increased Km values, modifying the promoters or other regulatory
elements that control the expression of one or more genes,
engineering the enzymes or the mRNA encoding these enzymes for a
decreased stability, misdirecting enzymes to cellular compartments
where they are less likely to interact with substrate and
intermediates, the use of interfering RNA, and the like.
[0050] As used herein, "aerobic fermentation" refers to growth in
the presence of oxygen.
[0051] As used herein, "anaerobic fermentation" refers to growth in
the absence of oxygen.
[0052] As used herein, the expression "end of fermentation" refers
to the stage of fermentation when the economic advantage of
continuing fermentation to produce a small amount of additional
alcohol is exceeded by the cost of continuing fermentation in terms
of fixed and variable costs. In a more general sense, "end of
fermentation" refers to the point where a fermentation will no
longer produce a significant amount of additional alcohol, i.e., no
more than about 1% additional alcohol, or no more substrate left
for further alcohol production.
[0053] As used, herein, a "glucoamylase unit (GAU)" is defined as
the amount of glucoamylase required to produce 1 g of glucose per
hour from soluble starch substrate (4% ds) under assay conditions
of 60.degree. C. and pH 4.2.
[0054] As used herein, a "spectrophotometric acid protease unit
(SAPU)" is the amount of protease activity that liberates one
micromole of tyrosine per minute from a casein substrate under
standard assay conditions.
[0055] As used herein, a "soluble starch unit (SSU)" is based on
the degree of hydrolysis of soluble potato starch substrate (4% DS)
by an aliquot of .alpha.-amylase at pH 4.5, 50.degree. C. The
reducing sugar content is measured using the DNS method as
described by Miller, G. L. ((1959) Anal. Chem. 31:426-28).
[0056] As used herein, the singular articles "a," "an" and "the"
encompass the plural referents unless the context clearly dictates
otherwise. All references cited herein are hereby incorporated by
reference in their entirety. The following abbreviations/acronyms
have the following meanings unless otherwise specified:
[0057] .degree. C. degrees Centigrade
[0058] AA .alpha.-amylase
[0059] AADH acetaldehyde dehydrogenases
[0060] ADH alcohol dehydrogenase
[0061] bp base pairs
[0062] DNA deoxyribonucleic acid
[0063] ds or DS dry solids
[0064] EC enzyme commission
[0065] EtOH ethanol
[0066] g or gm gram
[0067] g/L grams per liter
[0068] GA glucoamylase
[0069] GAU glucoamylase unit
[0070] HPLC high performance liquid chromatography
[0071] hr or h hour
[0072] kg kilogram
[0073] M molarp
[0074] mg milligram
[0075] min minute
[0076] mL or ml milliliter
[0077] mM millimolar
[0078] mRNA messenger RNA
[0079] N normal
[0080] nm nanometer
[0081] PCR polymerase chain reaction
[0082] PKL phosphoketolase
[0083] ppm parts per million
[0084] PTA phosphotransacetylase
[0085] SAPU spectrophotometric acid protease unit
[0086] SSU soluble starch unit
[0087] .DELTA. relating to a deletion
[0088] .mu.g microgram
[0089] .mu.L and .mu.l microliter
[0090] .mu.M micromolar
II. Overexpression of GIS1 in Yeast Increases Ethanol Production in
Yeast
[0091] GIS1 is a transcription factor involved in the regulation of
gene expression upon nutrient starvation. It recognizes and binds
to the post-diauxic-shift element 5'-T[AT]AGGGAT-3' (SEQ ID NO: 1)
in the promoter region. It can act as a transcriptional activator
(e.g., of stress genes like SSA3, HSP12 and HSP26) as well as a
repressor (e.g., of pyrophosphate phosphatase DPP1). GIS1 also acts
as a DNA damage-responsive transcriptional repressor of photolyase
PHR1. GIS1 and RPH1 have also been found to control a number of
genes involved in acetate and glycerol formation, metabolites that
have been implicated in aging.
[0092] It has now been discovered that GIS1 over-expression can
increase ethanol production by more than 1% in non-genetically
engineered yeast as well as engineered yeast harboring a
genetically engineered pathway. RNAseq analyses suggest that GIS1
gene is normally weakly expressed. 10-fold over-expression of GIS1
increases ethanol production in otherwise identical yeast by at
least 1%. The exemplified GIS1 polypeptide is from Saccharomyces
cerevisiae S288c (Genbank Accession No. NP_010381.1; SEQ ID NO: 5;
infra). Genbank lists numerous other polypeptide sequences with a
high percentage of amino acid sequence identity to SEQ ID NO: 5,
followed by a large number of other similar polypeptides. Most, if
not all, of these similar polypeptides are expected to produce
similar results in yeast cells.
[0093] In particular embodiments of the present compositions and
methods, the amino acid sequence of the GIS1 polypeptide that is
over-expressed in modified yeast cells has at least about 70%, at
least about 75%, at least about 80%, at least about 85%, at least
about 90%, at least about 91%, at least about 92%, at least about
93%, at least about 94%, at least about 95%, at least about 96%, at
least about 97%, at least about 98%, or even at least about 99%
identity, to SEQ ID NO: 5.
[0094] The increase in the amount of GIS1 produced by the modified
cells may be an increase of at least 20%, at least 30%, at least
40%, at least 50%, at least 70%, at least 100%, at least 150%, at
least 200%, at least 500%, at least 1,000%, or more, compared to
the amount of GIS1 produced by parental cells grown under the same
conditions. Alternatively, or additionally, the increase in the
amount of GIS1 produced by the modified cells may be at least
10-fold, at least 12 fold, or at least 15 fold, or at least
20-fold, or more, compared to the amount of GIS1 produced by
parental cells grown under the same conditions.
[0095] The increase in the strength of the promoter used to control
expression of the GIS1 produced by the modified cells may be at
least 10-fold, at least 12 fold, at least 15 fold, or at least
20-fold, or more, compared to strength of the native promoter
controlling GIS1 expression, based on the amount of mRNA
produced.
[0096] It is understood that relative promoter strength is not an
exact scalar value. It can strongly depend on culture medium,
fermentation time, temperature and other conditions. Values
obtained from RNAseq data collected over the time course of
fermentation in industrial medium are the most preferred, however,
experimental and/or literature data obtained under different
cultivation may also be used for recombinant promoter
selection.
[0097] Preferably, increased GIS1 expression is achieved by genetic
manipulation using sequence-specific molecular biology techniques,
as opposed to chemical mutagenesis, which is generally not targeted
to specific nucleic acid sequences. However, chemical mutagenesis
is not excluded as a method for making modified yeast cells.
[0098] In some embodiments, the present compositions and methods
involve introducing into yeast cells a nucleic acid capable of
directing the over-expression, or increased expression, of GIS1
polypeptide. Particular methods include but are not limited to (i)
introducing an exogenous expression cassette for producing the
polypeptide into a host cell, optionally in addition to an
endogenous expression cassette, (ii) substituting an exogenous
expression cassette with an endogenous cassette that allows the
production of an increased amount of the polypeptide, (iii)
modifying the promoter of an endogenous expression cassette to
increase expression, (iv) increase copy number of the same or
different cassettes for over-expression of GIS1 polypeptides,
and/or (v) modifying any aspect of the host cell to increase the
half-life of the polypeptide in the host cell.
[0099] In some embodiments, the parental cell that is modified
already includes an engineered pathway of interest, such as a PKL
pathway to increases ethanol production, or any other pathway to
increase alcohol production. In some embodiments, the parental cell
that is modified already includes a gene of interest, such as a
gene encoding a selectable marker, carbohydrate-processing enzyme,
or other polypeptide. In some embodiments, a gene of introduced is
subsequently introduced into the modified cells.
III. Modified Yeast Cells Overexpressing GIS1 and Harboring an
Exogenous PKL Pathway
[0100] Increased expression of GIS1 can be combined with expression
of genes of the PKL pathway to further increase the production
rate, and/or the maximum production levels, of ethanol in yeast
cells.
[0101] Engineered yeast cells having a heterologous PKL pathway
have been previously described in WO2015148272 (Miasnikov et al.).
These cells express heterologous phosphoketolase (PKL),
phosphotransacetylase (PTA) and acetylating acetyl dehydrogenase
(AADH), optionally with other enzymes, to channel carbon flux away
from the glycerol pathway and toward the synthesis of acetyl-CoA,
which is then converted to ethanol. Such modified cells are capable
of increased ethanol production in a fermentation process when
compared to otherwise-identical parent yeast cells.
IV. Overexpressing of GIS1 in Combination with Other Mutations that
Affect Methanol Production
[0102] In some embodiments, in addition to overexpressing GIS1,
optionally in combination with the presence of an exogenous PKL
pathway, the present modified yeast cells include additional
modifications that affect ethanol production.
[0103] The modified cells may further include mutations that result
in attenuation of the native glycerol biosynthesis pathway and/or
reuse glycerol pathway, which are known to increase alcohol
production. Methods for attenuation of the glycerol biosynthesis
pathway in yeast are known and include reduction or elimination of
endogenous NAD-dependent glycerol 3-phosphate dehydrogenase (GPD)
or glycerol phosphate phosphatase activity (GPP), for example by
disruption of one or more of the genes GPD1, GPD2, GPP1 and/or
GPP2. See, e.g., U.S. Pat. No. 9,175,270 (Elke et al.), U.S. Pat.
No. 8,795,998 (Pronk et al.) and U.S. Pat. No. 8,956,851 (Argyros
et al.). Methods to enhance the reuse glycerol pathway by over
expression of glycerol dehydrogenase (GCY1) and dihydroxyacetone
kinase (DAK1) to convert glycerol to dihydroxyacetone phosphate
(Zhang et al. (2013)J. Ind Microbiol. Biotechnol.
40:1153-1160).
[0104] The modified yeast may further feature increased acetyl-CoA
synthase (also referred to acetyl-CoA ligase) activity (EC 6.2.1.1)
to scavenge (i.e., capture) acetate produced by chemical or
enzymatic hydrolysis of acetyl-phosphate (or present in the culture
medium of the yeast for any other reason) and converts it to
Ac-CoA. This partially reduces the undesirable effect of acetate on
the growth of yeast cells and may further contribute to an
improvement in alcohol yield. Increasing acetyl-CoA synthase
activity may be accomplished by introducing a heterologous
acetyl-CoA synthase gene into cells, increasing the expression of
an endogenous acetyl-CoA synthase gene and the like.
[0105] In some embodiments the modified cells may further include a
heterologous gene encoding a protein with NADtdependent acetylating
acetaldehyde dehydrogenase (AADH) activity and/or a heterologous
gene encoding a pyruvate formate lyase (PFL). The introduction of
such genes in combination with attenuation of the glycerol pathway
is described, e.g., in U.S. Pat. No. 8,795,998 (Pronk et al.). In
some embodiments of the present compositions and methods the yeast
expressly lacks a heterologous gene(s) encoding an acetylating
acetaldehyde dehydrogenase, a pyruvate formate lyase or both.
[0106] In some embodiments, the present modified yeast cells may
further overexpress a sugar transporter-like (STL1) polypeptide to
increase the uptake of glycerol (see, e.g., Ferreira et al. (2005)
Mol Biol Cell 16:2068-76; Duskova et al. (2015) Mol Microbiol
97:541-59 and WO 2015023989 A1).
[0107] In some embodiments, the present modified yeast cells
further include a butanol biosynthetic pathway. In some
embodiments, the butanol biosynthetic pathway is an isobutanol
biosynthetic pathway. In some embodiments, the isobutanol
biosynthetic pathway comprises a polynucleotide encoding a
polypeptide that catalyzes a substrate to product conversion
selected from the group consisting of: (a) pyruvate to
acetolactate; (b) acetolactate to 2,3-dihydroxyisovalerate; (c)
2,3-dihydroxyisovalerate to 2-ketoisovalerate; (d)
2-ketoisovalerate to isobutyraldehyde; and (e) isobutyraldehyde to
isobutanol. In some embodiments, the isobutanol biosynthetic
pathway comprises polynucleotides encoding polypeptides having
acetolactate synthase, keto acid reductoisomerase, dihydroxy acid
dehydratase, ketoisovalerate decarboxylase, and alcohol
dehydrogenase activity.
[0108] In some embodiments, the modified yeast cells comprising a
butanol biosynthetic pathway further comprise a modification in a
polynucleotide encoding a polypeptide having pyruvate decarboxylase
activity. In some embodiments, the yeast cells comprise a deletion,
mutation, and/or substitution in an endogenous polynucleotide
encoding a polypeptide having pyruvate decarboxylase activity. In
some embodiments, the polypeptide having pyruvate decarboxylase
activity is selected from the group consisting of: PDC1, PDCS,
PDC6, and combinations thereof. In some embodiments, the yeast
cells further comprise a deletion, mutation, and/or substitution in
one or more endogenous polynucleotides encoding FRA2, ALD6, ADH1,
GPD2, BDH1, and YMR226C.
V. GIS1 Overexpression in Combination with Other Beneficial
Mutations
[0109] In some embodiments, in addition to overexpression of GIS1,
optionally in combination with other genetic modifications that
benefit alcohol production, the present modified yeast cells
further include any number of additional genes of interest encoding
proteins of interest, or of reducing the production, thereof.
Additional genes of interest may be introduced before, during, or
after genetic manipulations that result in the overexpression of
GIS1 polypeptides. Proteins of interest, include selectable
markers, carbohydrate-processing enzymes, and other
commercially-relevant polypeptides, including but not limited to an
enzyme selected from the group consisting of a dehydrogenase, a
transketolase, a phosphoketolase, a transladolase, an epimerase, a
phytase, a xylanase, a .beta.-glucanase, a phosphatase, a protease,
an .alpha.-amylase, a .beta.-amylase, a glucoamylase, a
pullulanase, an isoamylase, a cellulase, a trehalase, a lipase, a
pectinase, a polyesterase, a cutinase, an oxidase, a transferase, a
reductase, a hemicellulase, a mannanase, an esterase, an isomerase,
a pectinases, a lactase, a peroxidase and a laccase. Proteins of
interest may be secreted, glycosylated, and otherwise-modified.
VI. Use of the Modified Yeast for Increased Alcohol Production
[0110] The present yeast strains and methods of use, thereof, are
for increasing the production of alcohol in yeast. Such methods are
not limited to a particular fermentation process. The present
engineered yeast is expected to be a "drop-in" replacement for
convention yeast in any alcohol fermentation facility, whether
using raw starch hydrolysis, simultaneous saccharification and
fermentation, or other standard variations of conventional ethanol
production. While primarily intended for fuel alcohol production,
the present yeast can also be used for the production of potable
alcohol, including wine and beer.
VII. Yeast Cells Suitable for Modification
[0111] Yeasts are unicellular eukaryotic microorganisms classified
as members of the fungus kingdom and include organisms from the
phyla Ascomycota and Basidiomycota. Yeast that can be used for
alcohol production include, but are not limited to, Saccharomyces
spp., including S. cerevisiae, as well as Kluyveromyces, Lachancea
and Schizosaccharomyces spp. Numerous yeast strains are
commercially available, many of which have been selected or
genetically engineered for desired characteristics, such as high
alcohol production, rapid growth rate, and the like. Some yeast has
been genetically engineered to produce heterologous enzymes, such
as glucoamylase or .alpha.-amylase.
VII. Substrates and Products
[0112] Alcohol production from a number of carbohydrate substrates,
including but not limited to corn starch, sugar cane, cassava, and
molasses, is well known, as are innumerable variations and
improvements to enzymatic and chemical conditions and mechanical
processes. The present compositions and methods are believed to be
fully compatible with such substrates and conditions.
[0113] Alcohol fermentation products include organic compound
having a hydroxyl functional group (--OH) is bound to a carbon
atom. Exemplary alcohols include but are not limited to methanol,
ethanol, n-propanol, isopropanol, n-butanol, isobutanol,
n-pentanol, 2-pentanol, isopentanol, and higher alcohols. The most
commonly made fuel alcohols are ethanol, and butanol.
[0114] These and other aspects and embodiments of the present yeast
strains and methods will be apparent to the skilled person in view
of the present description.
EXAMPLES
[0115] The following examples are intended to further illustrate,
but not limit, the described compositions and methods.
Example 1. Materials and Methods
[0116] The following protocols were employed unless otherwise
specified.
Liquefact Preparation
[0117] Liquefact (Ground corn slurry) was prepared by adding 600
ppm of urea, 0.124 SAPU/g ds FERMGEN.TM. 2.5.times. (acid fungal
protease), 0.33 GAU/g ds glucoamylase (a variant Trichoderma
glucoamylase) and 1.46 SSU/g ds GC626 (Aspergillus
.alpha.-amylase), adjusted to a pH of 4.8.
AnKom Assays
[0118] 300 .mu.L of concentrated yeast overnight culture was added
to each of a number ANKOM bottles filled with 30 g prepared
liquefact for a final OD of 0.3. The bottles were then incubated at
32.degree. C. with shaking (150 RPM) for 65 hours.
HPLC Analysis
[0119] Samples from serum vial and AnKom experiments were collected
in Eppendorf tubes by centrifugation for 12 minutes at 14,000 RPM.
The supernatants were filtered with 0.2 .mu.M PTFE filters and then
used for HPLC (Agilent Technologies 1200 series) analysis with the
following conditions: Bio-Rad Aminex HPX-87H columns, running
temperature of 55.degree. C. 0.6 ml/min isocratic flow 0.01 N
H.sub.2SO.sub.4, 2.5 .mu.L injection volume. Calibration standards
were used for quantification of the of acetate, ethanol, glycerol,
and glucose. Samples from shake flasks experiments were collected
in Eppendorf tubes by centrifugation for 15 minutes at 14,000 RPM.
The supernatants were diluted by a factor of 11 using 5 mM
H.sub.2SO.sub.4 and incubated for 5 min at 95.degree. C. Following
cooling, samples were filtered with 0.2 .mu.M Corning FiltrEX
CLS3505 filters and then used for HPLC analysis. 10 .mu.l was
injected into an Agilent 1200 series HPLC equipped with a
refractive index detector. The separation column used was a
Phenomenex Rezex-RFQ Fast Acid H+ (8%) column. The mobile phase was
5 mM H.sub.2SO.sub.4, and the flow rate was 1.0 mL/min at
85.degree. C. HPLC Calibration Standard Mix from Bion Analytical
was used for quantification of the of acetate, ethanol, glycerol,
and glucose. Unless otherwise specified, all values are expressed
in g/L.
Example 2. Over-Expression of Codon-Optimized GIS1 in Yeast
[0120] GIS1, encoded by gene YDR096W of Saccharomyces cerevisiae
S288c, was codon optimized and synthesized to generate the
artificial gene, "GIS1s," shown as SEQ ID NO: 2:
TABLE-US-00002 ATGGAAATCAAGCCAGTCGAAGTTATCGACGGTGTTCCAGTCTTCAAGCCA
TCTATGATGGAATTTGCCAATTTTCAATACTTCATTGACGAAATCACCAAG
TTTGGTATCGAAAACGGTATTGTCAAGGTTATTCCTCCCAAGGAATGGCTG
GAATTGTTGGAAGGTTCTCCACCTGCTGAATCCTTGAAGACTATCCAACTA
GATTCTCCAATTCAACAGCAAGCCAAGAGATGGGACAAACACGAAAACGGT
GTCTTTTCCATCGAAAACGAATACGACAACAAGTCTTACAACTTGACACAA
TGGAAGAATTTGGCTGAATCCTTGGATTCTAGAATCAGTCAAGGTGACTTC
AACGACAAGACCTTAAAGGAAAACTGCAGAGTCGATTCTCAACAGGATTGT
TACGATTTGGCTCAATTACAAATCTTGGAATCCGACTTCTGGAAGACCATT
GCCTTTTCCAAGCCATTCTACGCTGTTGACGAAAACTCTTCCATCTTCCCA
TACGACTTGACTTTATGGAACTTGAACAATTTGCCAGATTCTATCAACTCC
AGCAACAGACGTTTGCTAACTGGTCAATCCAAGTGTATCTTTCCATGGCAC
TTGGACGAACAAAACAAGTGTTCTATCAACTACTTGCACTTCGGTGCTCCA
AAGCAATGGTACTCCATTCCATCTGCCAACACCGATCAATTCTTGAAGATC
CTATCCAAGGAACCATCAAGCAACAAGGAAAATTGTCCAGCTTTCATCCGT
CATCAAAACATCATTACTTCTCCAGACTTTTTGAGAAAGAACAATATCAAG
TTCAACAGAGTTGTCCATTTCAACATGAATTTATCATTACCTTTCCTTACT
GTATGTACTCCGGTTTCAACTACGGTTACAACTTTGGCGAATCTATCGAGT
TCATCTTAGATCAGCAAGCTGTTGTCAGAAAGCAACCATTGAAGTGTGGTT
GCGGCAACAAGAAAGAAGAGAGAAAGTCTGGTCCATTTTCCAACTTGTCTT
ACGACTCCAACGAAAGCGAACAACGTGGTTCTATTACCGACAACGACAATG
ATTTGTTTCAAAAGGTCAGATCCTTCGACGAATTGCTAAACCACTCCTCTC
AAGAATTGCAAAACTTGGAAGACAACAAGAATCCATTGTTTTCCAACATCA
ATATGAACAGACCACAAAGCTCCTCTTTGAGGTCTACTACACCAAACGGTG
TCAACCAATTCTTGAACATGAATCAAACTACCATCAGCAGAATTTCCTCTC
CATTGTTATCAAGAATGATGGACTTGTCCAACATCGTGGAACCAACCTTGG
ACGATCCTGGTTCCAAGTTCAAGAGAAAGGTTTTGACTCCACAATTACCAC
AAATGAACATTCCATCCAACTCTTCCAACTTTGGTACTCCTTCTTTGACCA
ATACAAACTCCTTGCTATCAAACATCACTGCTACATCTACCAATCCATCCA
CCACTACCAACGGCTCTCAAAACCACAACAATGTCAACGCCAATGGTATCA
ACACCTCTGCTGCCGCTTCCATCAACAATAACATTTCCTCTACCAACAATT
CTGCCAATAACAGCTCTTCCAACAATAACGTTTCTACTGTTCCATCTTCCA
TGATGCACTCTTCCACCTTGAATGGTACTTCTGGTTTGGGTGGCGACAACG
ATGACAACATGTTAGCTTTGAGCCTAGCTACCTTGGCCAACAGTGCTACTG
CTTCTCCAAGATTGACCTTACCACCTTTGTCTTCACCAATGAATCCCAACG
GTCACACTTCCTACAACGGTAACATGATGAACAATAACTCTGGTAACGGTT
CCAACGGTAGCAACTCTTACTCCAATGGTGTCACCACTGCTGCCGCTACCA
CTACATCTGCTCCACACAACTTGTCCATCGTTTCTCCAAACCCTACCTACA
GTCCAAATCCCTTGTCTCTATACTTGACCAACTCCAAGAATCCATTGAACT
CTGGTTTGGCTCCATTATCTCCTTCCACTTCTAACATTCCATTCTTGAAGA
GAAACAATGTCGTTACCCTAAACATCTCCAGAGAAGCCTCCAAGTCTCCAA
TCTCTTCCTTTGTCAACGACTACCGTTCTCCATTGGGTGTTTCCAATCCAT
TGATGTACTCTTCCACTATCAACGATTACTCCAACGGTACTGGTATCCGTC
AAAACAGCAACAACATCAATCCATTGGACGCTGGTCCATCTTTTTCTCCTT
TGCACAAGAAACCAAAGATTTTGAACGGCAATGACAACTCCAATTTGGACA
GCAACAATTTCGATTACTCTTTTACTGGTAACAAGCAAGAATCCAATCCAT
CTATCTTGAACAACAATACCAACAATAACGACAACTACAGAACTTCTTCCA
TGAACAACAATGGTAACAATTACCAAGCTCACTCTTCCAAGTTTGGTGAAA
ACGAAGTCATCATGTCCGATCACGGTAAGATTTACATCTGTAGAGAATGTA
ACAGACAATTTTCCTCTGGTCACCATCTAACCAGACACAAAAAGTCAGTTC
ATTCTGGTGAAAAGCCACACTCTTGTCCAAGATGTGGTAAAAGATTCAAGA
GAAGAGATCATGTCTTGCAACACTTGAACAAGAAAATCCCATGCACTCAAG
AAATGGAAAACACCAAGTTGGCTGAATCTTAA
[0121] The ADR1 promoter (represented by SEQ ID NO: 3) was
operably-linked to the 5'-end of the codon-optimized GIS1s coding
sequence to control expression:
TABLE-US-00003 AATAGAGTATGATTATTTTTTTTATATTTTTTTTTTTTGGAAAACAAAATT
CTTATAGTAAAGTAAGGAATAGTAGCAGAATATTTTTCTGAAGTGTTTATA
ATAAAGGGAGAACCGGGAAAGTAGCAAAATGATTGGTTAATTTATGCAAAT
CAATCTTATACTTCCAACGAATAAGAGGGAGTATATCAAAACAGAGTAACA
ATAAACTTTTGCTATGACACCTTTTCTTTCTTTCAAAGATAAAAGAATAAG
GCTTTTCTATAGTAGTCGAGCAAATGTTGGAATAAGGAGGTATGGAATTTT
GAAAATAGCCTGAGAAATTCAGATCAATGATATATAACTGTTGTCTTCAAA
AGGTCTTCAAGGGAAGAAAATTTCCGATCGGAAGTCCAGAATAAATACCAC
AAGTATAGCACAATAAACAACAGCACAACAACAATAATAACAACACCTGTA
GCGAAAACGTTTTCCTATTTTTAAGGCGTTGTTCTTTGAAAACCTGTGGCG
AAGTAAAACATGAAAACAAATGAAAACACCGCCAAACCAAGAAAAGAACAA
CGAAAAAATATCACTTTTATTTAGTTCACAACGGCTAACTATCGACGTTCA
CCCTTCCTCAGTCTATCACATCGTCCTTAGCTCGAACAACGCCGATAGGCA
TCAAGTTACATTGAGCTTTACTGCACGTTCCCGCATGATGCCATTGACTAG
GGCCCGCCCTTTCGGCAATCATTCTAGCATGTTCCGCATGTTC
[0122] The Eno2 terminator (YHR174W locus; SEQ ID NO: 4) was
operably linked to the 3'-end of the codon-optimized GIS1s coding
sequence to complete the ADR1Pro::GIS1ss::Ebo2Ter expression
cassette:
TABLE-US-00004 GTGCACCTTTTTTTTCTCCTTCCAGTGCATTATGCAATAGACAGCACGAGT
CTTTGAAAAAGTAACTTATAAAACTGTATCAATTTTTAAACCTAAATAGAT
TCATAAACTATTCGTTAATATAAAGTGTTCTAAACTATGATGAAAAAATAA
GCAGAAAAGACTAATAATTCTTAGTTAAAAGCACTTTACAACTTGTCACCG
TGGTGGAAGTTTTCACCG
[0123] The amino acid sequence of the GIS1 polypeptide from
Saccharomyces cerevisiae S288c (Genbank Accession No. NP_010381.1)
is shown, below, as SEQ ID NO: 5:
TABLE-US-00005 MEIKPVEVIDGVPVFKPSMMEFANFQYFIDEITKFGIENGIVKVIPPKEWL
ELLEGSPPAESLKTIQLDSPIQQQAKRWDKHENGVFSIENEYDNKSYNLTQ
WKNLAESLDSRISQGDFNDKTLKENCRVDSQQDCYDLAQLQILESDFWKTI
AFSKPFYAVDENSSIFPYDLTLWNLNNLPDSINSSNRRLLTGQSKCIFPWH
LDEQNKCSINYLHFGAPKQWYSIPSANTDQFLKILSKEPSSNKENCPAFIR
HQNIITSPDFLRKNNIKFNRVVQFQHEFIITFPYCMYSGFNYGYNFGESIE
FILDQQAVVRKQPLKCGCGNKKEERKSGPFSNLSYDSNESEQRGSITDNDN
DLFQKVRSFDELLNHSSQELQNLEDNKNPLFSNINMNRPQSSSLRSTTPNG
VNQFLNMNQTTISRISSPLLSRMMDLSNIVEPTLDDPGSKFKRKVLTPQLP
QMNIPSNSSNFGTPSLTNTNSLLSNITATSTNPSTTTNGSQNHNNVNANGI
NTSAAASINNNISSTNNSANNSSSNNNVSTVPSSMMHSSTLNGTSGLGGDN
DDNMLALSLATLANSATASPRLTLPPLSSPMNPNGHTSYNGNMMNNNSGNG
SNGSNSYSNGVTTAAATTTSAPHNLSIVSPNPTYSPNPLSLYLTNSKNPLN
SGLAPLSPSTSNIPFLKRNNVVTLNISREASKSPISSFVNDYRSPLGVSNP
LMYSSTINDYSNGTGIRQNSNNINPLDAGPSFSPLHKKPKILNGNDNSNLD
SNNFDYSFTGNKQESNPSILNNNTNNNDNYRTSSMNNNGNNYQAHSSKFGE
NEVIMSDHGKIYICRECNRQFSSGHHLTRHKKSVHSGEKPHSCPRCGKRFK
RRDHVLQHLNKKIPCTQEMENTKLAES
[0124] This expression cassette was amplified using primers with
appropriate flanking sequences to promote insertion at the PCT7
site (YHR076W) and transformed into either (i) FERMAXTM Gold
(Martrex Inc., Minnesota, USA; herein abbreviated, "FG"), a
well-known fermentation yeast used in the grain ethanol industry,
or (ii) FG-PKL, engineered FG yeast having a heterologous
phosphoketolase (PKL) pathway involving the expression of
phosphoketolase (PKL), phosphotransacetylase (PTA) and acetylating
acetyl dehydrogenase (AADH) as described in WO2015148272 (Miasnikov
et al.). The expected insertion of the GIS1 expression cassette
into the two parental strains was confirmed by PCR.
Example 3. Alcohol Production using Yeast that Over-Express
GIS1s
[0125] Strains over-expressing GIS1 were tested in an Ankom assay,
containing 30 g liquefact. Fermentations were performed at
32.degree. C. for 55 hours. Samples from the end of fermentation
were analyzed by HPLC. The results are summarized in Tables 1 and
2.
TABLE-US-00006 TABLE 1 HPLC results from FG and FG-GIS1s strains
Glycerol Acetate Glucose Ethanol Ethanol Strain features (g/L)
(g/L) (g/L) (g/L) increase (%) FG 13.06 0.91 0.33 143.44 -0-
FG-GIS1s 12.99 0.85 0.40 145.42 1.4
TABLE-US-00007 TABLE 2 HPLC results from FG-PKL and FG-PKL-GIS1s
Glycerol Acetate Glucose Ethanol Ethanol Strain features (g/L)
(g/L) (g/L) (g/L) increase (%) FG-PKL 12.53 1.55 0.70 147.27 -0-
FG-PKL-GIS1s 12.01 1.72 0.26 148.90 1.1
[0126] Over-expression of GIS1 resulted in 1.4% increase of ethanol
production in FG yeast, which is recognized as a robust,
high-ethanol-producing yeast for the fuel ethanol industry, while
not being a genetically engineered organism. Over-expression of
GIS1s resulted 1.1% increase of ethanol production in FG yeast
engineered to have an exogenous PKL pathway. These results again
demonstrate that GIS1 over-expression is beneficial for increasing
ethanol production.
Sequence CWU 1
1
519DNAArtificial SequenceArtificial element 1tatagggat
922685DNASaccharomyces cerevisiae 2atggaaatca agccagtcga agttatcgac
ggtgttccag tcttcaagcc atctatgatg 60gaatttgcca attttcaata cttcattgac
gaaatcacca agtttggtat cgaaaacggt 120attgtcaagg ttattcctcc
caaggaatgg ctggaattgt tggaaggttc tccacctgct 180gaatccttga
agactatcca actagattct ccaattcaac agcaagccaa gagatgggac
240aaacacgaaa acggtgtctt ttccatcgaa aacgaatacg acaacaagtc
ttacaacttg 300acacaatgga agaatttggc tgaatccttg gattctagaa
tcagtcaagg tgacttcaac 360gacaagacct taaaggaaaa ctgcagagtc
gattctcaac aggattgtta cgatttggct 420caattacaaa tcttggaatc
cgacttctgg aagaccattg ccttttccaa gccattctac 480gctgttgacg
aaaactcttc catcttccca tacgacttga ctttatggaa cttgaacaat
540ttgccagatt ctatcaactc cagcaacaga cgtttgctaa ctggtcaatc
caagtgtatc 600tttccatggc acttggacga acaaaacaag tgttctatca
actacttgca cttcggtgct 660ccaaagcaat ggtactccat tccatctgcc
aacaccgatc aattcttgaa gatcctatcc 720aaggaaccat caagcaacaa
ggaaaattgt ccagctttca tccgtcatca aaacatcatt 780acttctccag
actttttgag aaagaacaat atcaagttca acagagttgt ccaatttcaa
840catgaattta tcattacctt tccttactgt atgtactccg gtttcaacta
cggttacaac 900tttggcgaat ctatcgagtt catcttagat cagcaagctg
ttgtcagaaa gcaaccattg 960aagtgtggtt gcggcaacaa gaaagaagag
agaaagtctg gtccattttc caacttgtct 1020tacgactcca acgaaagcga
acaacgtggt tctattaccg acaacgacaa tgatttgttt 1080caaaaggtca
gatccttcga cgaattgcta aaccactcct ctcaagaatt gcaaaacttg
1140gaagacaaca agaatccatt gttttccaac atcaatatga acagaccaca
aagctcctct 1200ttgaggtcta ctacaccaaa cggtgtcaac caattcttga
acatgaatca aactaccatc 1260agcagaattt cctctccatt gttatcaaga
atgatggact tgtccaacat cgtggaacca 1320accttggacg atcctggttc
caagttcaag agaaaggttt tgactccaca attaccacaa 1380atgaacattc
catccaactc ttccaacttt ggtactcctt ctttgaccaa tacaaactcc
1440ttgctatcaa acatcactgc tacatctacc aatccatcca ccactaccaa
cggctctcaa 1500aaccacaaca atgtcaacgc caatggtatc aacacctctg
ctgccgcttc catcaacaat 1560aacatttcct ctaccaacaa ttctgccaat
aacagctctt ccaacaataa cgtttctact 1620gttccatctt ccatgatgca
ctcttccacc ttgaatggta cttctggttt gggtggcgac 1680aacgatgaca
acatgttagc tttgagccta gctaccttgg ccaacagtgc tactgcttct
1740ccaagattga ccttaccacc tttgtcttca ccaatgaatc ccaacggtca
cacttcctac 1800aacggtaaca tgatgaacaa taactctggt aacggttcca
acggtagcaa ctcttactcc 1860aatggtgtca ccactgctgc cgctaccact
acatctgctc cacacaactt gtccatcgtt 1920tctccaaacc ctacctacag
tccaaatccc ttgtctctat acttgaccaa ctccaagaat 1980ccattgaact
ctggtttggc tccattatct ccttccactt ctaacattcc attcttgaag
2040agaaacaatg tcgttaccct aaacatctcc agagaagcct ccaagtctcc
aatctcttcc 2100tttgtcaacg actaccgttc tccattgggt gtttccaatc
cattgatgta ctcttccact 2160atcaacgatt actccaacgg tactggtatc
cgtcaaaaca gcaacaacat caatccattg 2220gacgctggtc catctttttc
tcctttgcac aagaaaccaa agattttgaa cggcaatgac 2280aactccaatt
tggacagcaa caatttcgat tactctttta ctggtaacaa gcaagaatcc
2340aatccatcta tcttgaacaa caataccaac aataacgaca actacagaac
ttcttccatg 2400aacaacaatg gtaacaatta ccaagctcac tcttccaagt
ttggtgaaaa cgaagtcatc 2460atgtccgatc acggtaagat ttacatctgt
agagaatgta acagacaatt ttcctctggt 2520caccatctaa ccagacacaa
aaagtcagtt cattctggtg aaaagccaca ctcttgtcca 2580agatgtggta
aaagattcaa gagaagagat catgtcttgc aacacttgaa caagaaaatc
2640ccatgcactc aagaaatgga aaacaccaag ttggctgaat cttaa
26853757DNAArtificial SequenceArtificial promoter 3aatagagtat
gattattttt tttatatttt ttttttttgg aaaacaaaat tcttatagta 60aagtaaggaa
tagtagcaga atatttttct gaagtgttta taataaaggg agaaccggga
120aagtagcaaa atgattggtt aatttatgca aatcaatctt atacttccaa
cgaataagag 180ggagtatatc aaaacagagt aacaataaac ttttgctatg
acaccttttc tttctttcaa 240agataaaaga ataaggcttt tctatagtag
tcgagcaaat gttggaataa ggaggtatgg 300aattttgaaa atagcctgag
aaattcagat caatgatata taactgttgt cttcaaaagg 360tcttcaaggg
aagaaaattt ccgatcggaa gtccagaata aataccacaa gtatagcaca
420ataaacaaca gcacaacaac aataataaca acacctgtag cgaaaacgtt
ttcctatttt 480taaggcgttg ttctttgaaa acctgtggcg aagtaaaaca
tgaaaacaaa tgaaaacacc 540gccaaaccaa gaaaagaaca acgaaaaaat
atcactttta tttagttcac aacggctaac 600tatcgacgtt cacccttcct
cagtctatca catcgtcctt agctcgaaca acgccgatag 660gcatcaagtt
acattgagct ttactgcacg ttcccgcatg atgccattga ctagggcccg
720ccctttcggc aatcattcta gcatgttccg catgttc 7574222DNAArtificial
SequenceArtificial terminator 4gtgcaccttt tttttctcct tccagtgcat
tatgcaatag acagcacgag tctttgaaaa 60agtaacttat aaaactgtat caatttttaa
acctaaatag attcataaac tattcgttaa 120tataaagtgt tctaaactat
gatgaaaaaa taagcagaaa agactaataa ttcttagtta 180aaagcacttt
acaacttgtc accgtggtgg aagttttcac cg 2225894PRTSaccharomyces
cerevisiae 5Met Glu Ile Lys Pro Val Glu Val Ile Asp Gly Val Pro Val
Phe Lys1 5 10 15Pro Ser Met Met Glu Phe Ala Asn Phe Gln Tyr Phe Ile
Asp Glu Ile 20 25 30Thr Lys Phe Gly Ile Glu Asn Gly Ile Val Lys Val
Ile Pro Pro Lys 35 40 45Glu Trp Leu Glu Leu Leu Glu Gly Ser Pro Pro
Ala Glu Ser Leu Lys 50 55 60Thr Ile Gln Leu Asp Ser Pro Ile Gln Gln
Gln Ala Lys Arg Trp Asp65 70 75 80Lys His Glu Asn Gly Val Phe Ser
Ile Glu Asn Glu Tyr Asp Asn Lys 85 90 95Ser Tyr Asn Leu Thr Gln Trp
Lys Asn Leu Ala Glu Ser Leu Asp Ser 100 105 110Arg Ile Ser Gln Gly
Asp Phe Asn Asp Lys Thr Leu Lys Glu Asn Cys 115 120 125Arg Val Asp
Ser Gln Gln Asp Cys Tyr Asp Leu Ala Gln Leu Gln Ile 130 135 140Leu
Glu Ser Asp Phe Trp Lys Thr Ile Ala Phe Ser Lys Pro Phe Tyr145 150
155 160Ala Val Asp Glu Asn Ser Ser Ile Phe Pro Tyr Asp Leu Thr Leu
Trp 165 170 175Asn Leu Asn Asn Leu Pro Asp Ser Ile Asn Ser Ser Asn
Arg Arg Leu 180 185 190Leu Thr Gly Gln Ser Lys Cys Ile Phe Pro Trp
His Leu Asp Glu Gln 195 200 205Asn Lys Cys Ser Ile Asn Tyr Leu His
Phe Gly Ala Pro Lys Gln Trp 210 215 220Tyr Ser Ile Pro Ser Ala Asn
Thr Asp Gln Phe Leu Lys Ile Leu Ser225 230 235 240Lys Glu Pro Ser
Ser Asn Lys Glu Asn Cys Pro Ala Phe Ile Arg His 245 250 255Gln Asn
Ile Ile Thr Ser Pro Asp Phe Leu Arg Lys Asn Asn Ile Lys 260 265
270Phe Asn Arg Val Val Gln Phe Gln His Glu Phe Ile Ile Thr Phe Pro
275 280 285Tyr Cys Met Tyr Ser Gly Phe Asn Tyr Gly Tyr Asn Phe Gly
Glu Ser 290 295 300Ile Glu Phe Ile Leu Asp Gln Gln Ala Val Val Arg
Lys Gln Pro Leu305 310 315 320Lys Cys Gly Cys Gly Asn Lys Lys Glu
Glu Arg Lys Ser Gly Pro Phe 325 330 335Ser Asn Leu Ser Tyr Asp Ser
Asn Glu Ser Glu Gln Arg Gly Ser Ile 340 345 350Thr Asp Asn Asp Asn
Asp Leu Phe Gln Lys Val Arg Ser Phe Asp Glu 355 360 365Leu Leu Asn
His Ser Ser Gln Glu Leu Gln Asn Leu Glu Asp Asn Lys 370 375 380Asn
Pro Leu Phe Ser Asn Ile Asn Met Asn Arg Pro Gln Ser Ser Ser385 390
395 400Leu Arg Ser Thr Thr Pro Asn Gly Val Asn Gln Phe Leu Asn Met
Asn 405 410 415Gln Thr Thr Ile Ser Arg Ile Ser Ser Pro Leu Leu Ser
Arg Met Met 420 425 430Asp Leu Ser Asn Ile Val Glu Pro Thr Leu Asp
Asp Pro Gly Ser Lys 435 440 445Phe Lys Arg Lys Val Leu Thr Pro Gln
Leu Pro Gln Met Asn Ile Pro 450 455 460Ser Asn Ser Ser Asn Phe Gly
Thr Pro Ser Leu Thr Asn Thr Asn Ser465 470 475 480Leu Leu Ser Asn
Ile Thr Ala Thr Ser Thr Asn Pro Ser Thr Thr Thr 485 490 495Asn Gly
Ser Gln Asn His Asn Asn Val Asn Ala Asn Gly Ile Asn Thr 500 505
510Ser Ala Ala Ala Ser Ile Asn Asn Asn Ile Ser Ser Thr Asn Asn Ser
515 520 525Ala Asn Asn Ser Ser Ser Asn Asn Asn Val Ser Thr Val Pro
Ser Ser 530 535 540Met Met His Ser Ser Thr Leu Asn Gly Thr Ser Gly
Leu Gly Gly Asp545 550 555 560Asn Asp Asp Asn Met Leu Ala Leu Ser
Leu Ala Thr Leu Ala Asn Ser 565 570 575Ala Thr Ala Ser Pro Arg Leu
Thr Leu Pro Pro Leu Ser Ser Pro Met 580 585 590Asn Pro Asn Gly His
Thr Ser Tyr Asn Gly Asn Met Met Asn Asn Asn 595 600 605Ser Gly Asn
Gly Ser Asn Gly Ser Asn Ser Tyr Ser Asn Gly Val Thr 610 615 620Thr
Ala Ala Ala Thr Thr Thr Ser Ala Pro His Asn Leu Ser Ile Val625 630
635 640Ser Pro Asn Pro Thr Tyr Ser Pro Asn Pro Leu Ser Leu Tyr Leu
Thr 645 650 655Asn Ser Lys Asn Pro Leu Asn Ser Gly Leu Ala Pro Leu
Ser Pro Ser 660 665 670Thr Ser Asn Ile Pro Phe Leu Lys Arg Asn Asn
Val Val Thr Leu Asn 675 680 685Ile Ser Arg Glu Ala Ser Lys Ser Pro
Ile Ser Ser Phe Val Asn Asp 690 695 700Tyr Arg Ser Pro Leu Gly Val
Ser Asn Pro Leu Met Tyr Ser Ser Thr705 710 715 720Ile Asn Asp Tyr
Ser Asn Gly Thr Gly Ile Arg Gln Asn Ser Asn Asn 725 730 735Ile Asn
Pro Leu Asp Ala Gly Pro Ser Phe Ser Pro Leu His Lys Lys 740 745
750Pro Lys Ile Leu Asn Gly Asn Asp Asn Ser Asn Leu Asp Ser Asn Asn
755 760 765Phe Asp Tyr Ser Phe Thr Gly Asn Lys Gln Glu Ser Asn Pro
Ser Ile 770 775 780Leu Asn Asn Asn Thr Asn Asn Asn Asp Asn Tyr Arg
Thr Ser Ser Met785 790 795 800Asn Asn Asn Gly Asn Asn Tyr Gln Ala
His Ser Ser Lys Phe Gly Glu 805 810 815Asn Glu Val Ile Met Ser Asp
His Gly Lys Ile Tyr Ile Cys Arg Glu 820 825 830Cys Asn Arg Gln Phe
Ser Ser Gly His His Leu Thr Arg His Lys Lys 835 840 845Ser Val His
Ser Gly Glu Lys Pro His Ser Cys Pro Arg Cys Gly Lys 850 855 860Arg
Phe Lys Arg Arg Asp His Val Leu Gln His Leu Asn Lys Lys Ile865 870
875 880Pro Cys Thr Gln Glu Met Glu Asn Thr Lys Leu Ala Glu Ser 885
890
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